Intracellular bacterial pathogens, that is bacterial pathogens that inhabit another cell or microorganism, are a major cause of human morbidity and mortality. Evading hostile intracellular environments is one of the ways pathogens can live within a host cell, grow symbiotically or parasitically within host cells, and yet not be killed or inhibited by the host cell. These parasites have developed ways of interacting and overcoming the host cells natural defense mechanisms.
Legionella pneumophila, a bacterium known to cause Legionnaire's Disease and Pontiac fever in humans is a parasite of this type. While the Legionella cells can be killed readily if exposed to certain chemical agents and antibiotics, Legionella can also be found engulfed (phagocitized) within certain protozoa hosts. Legionella are often found in biofilms adsorbed to solid surfaces in, for example, water distribution systems, cooling towers, showers, aquaria, sprinklers, spas, and cleaning baths. Protozoa are natural grazers on surfaces and engulf and digest bacteria as part of their natural life cycle. In most cases, the protozoa digest these bacteria through the use of digestive enzymes in their phagosomes (digestive vacules). In the case of Legionella, however, this is not the case. The protozoa are not readily capable of degrading the engulfed Legionella cells, and in fact the Legionella grow and increase their numbers while protected within protozoa phagosomes. Legionellosis in humans can be contracted by breathing Legionella aerosols containing either the free-living bacterial cells or by inhaling aerosols of Legionella concentrated within susceptible protozoa. A Legionella control agent, therefore, must be capable of killing free-living Legionella, Legionella within protozoa, or the protozoa themselves. The agents described in this invention are capable of killing the free-living Legionella and the host protozoa. Two protozoa species capable of harboring infectious Legionella are Acanthamoeba and Tetrahymena. 
In order to effectively control Legionella, in addition to killing the free living or protozoa an additional factor must be taken into account. Certain protozoa, particularly amoeboid forms, have evolved mechanisms for surviving in hostile environments. Examples of hostile environments are high temperature, desiccation, presence of chemical agents/antibiotics, lack of food sources, etc. Upon encountering a hostile environment, these protozoa revert to a cyst form that is very difficult to kill. The cyst form becomes much less susceptible to chemical agents that readily kill the same organism when it is in non-cyst (trophozoite) form. Introduction of a chemical control agent to eliminate Acanthamoeba can actually provide the hostile environment to which the protozoa responds by reverting to a cyst form, thereby rendering it invulnerable to the chemical agent. When the cyst contains the pathogen Legionella, the chemical agent can no longer reach the engulfed bacteria, and the chemical treatment is rendered ineffective. As an example, chlorination or bleach is considered essential to control Legionella in water distribution systems. Exposed Legionella are readily killed by low levels of free chlorine (0.2-0.5 μg/ml).
Infective Legionella can also be contained in Acanthamoeba phagosomes if those protozoa are present. The Acanthamoeba, sensing the chlorine presence, reverts to a cyst form, inadvertently preserving and protecting the Legionella parasites engulfed within it. The Acanthamoeba cysts treated with >500 times (>100 μg/ml “free” chlorine) the concentration needed to kill the trophozoite forms are not killed in the cyst form. The cysts can revert to the active trophozoite form upon removal of the oxidant.
Currently there are no known cyst deactivating agents in commercial use at this time. Although control agents or biocides which effectively kill or treat the Legionella bacteria are known, there is no method currently in use which provides for the means to effectively introduce the biocides or control agents into the water systems where the Legionella bacteria and Legionella harboring protozoa and cysts reside. Control agents that kill the Legionella harboring protozoa and protozoan cysts provide a much needed additional tool to safeguard the health of workers and the public against the respiratory pneumonias which can result from inhalation of Legionella or Legionella-containing protozoa cysts. For example, U.S. Pat. No. 6,579,859 discloses the use of phosphonium salts of the general formula (R1)3P+R2.X− wherein R1 is an alkyl group of from 1 to 8 carbon atoms, R2 is an n-alkyl group giving 8 to 20 carbon atoms and X is an anion consisting of a halide, sulfate, nitrate, nitrite, etc.
US patent publication no. 2005/002710 teaches the exposure of the protozoa to quaternary ammonium salts, while US patent publication no. 2005/0080142 discloses the use of guanidine or biguanidine salts to control Legionella type bacteria in the free-living state as well as when engulfed in the trophozoite form or Acanthamoeba in cyst form.
However, the method of introducing these agents to the Legionella bacteria has been a barrier, particularly under actually working conditions. Therefore, a need still exists for a means to take the known biocide agents, such as those cited above, and put them in contact with the Legionella bacteria in a way that is efficient and effective, and will be of commercial use.